Labeled oligonucleotides find utility in a number of applications, including DNA sequencing, diagnostic detection or quantitation and forensic science. Typically, the labeled oligonucleotide is allowed to hybridize or anneal with nucleic acid present in the sample and the presence or absence of label is detected following separation steps.
Many mechanisms and schemes have been used to introduce labels into oligonucleotides. For a comprehensive review of these methods see Goodchild, Bioconjugate Chemistry, 1(3):165-187 (1990). According to Goodchild, past researchers have labeled oligonucleotides at both internal and terminal locations; by enzymatic and chemical synthetic means; and utilizing a single or many marker molecules per oligonucleotide. Methods involving incorporation of marker moieties at internal locations in the oligonucleotides are generally less preferable due to their less predictable hybridization behavior. For this reason, end labeled oligonucleotides are often preferred, particularly for automated detection systems. Similarly, methods for incorporating multiple label moieties into an oligonucleotide are less preferred for stoichiometric reasons. For some applications, it may not be critical that the labeled oligonucleotides are poorly characterized in terms of exact positioning and number of label molecules. However, for automated diagnostic detection, it is desireable that each oligonucleotide have a single marker moiety, preferably a haptenic "hook", at an end location.
Methods for placing a single marker, or hapten capable of reacting with an antibody or other specific binding member, at a terminal position on an oligonucleotide have been described in the literature. Most commonly, a linker member containing a primary amine or other nucleophilic group is attached at the 3' or 5' terminus to enable conjugation to one of numerous electrophilic detectable markers. Alternatively, terminal deoxynucleotidyl transferase, ligase and phosphoramidite chemistry have been used to attach direct labels or reactive linkers to oligonucleotides.
For example, Kempe, et al., Nucleic Acids Research 13(1):45-57 (1985) describe methods for post-synthesis biotinylation of 3' termini. A first method involves oxidation of the 3' hydroxyl to a 3' aldehyde, followed by condensation with alkyl diamines to provide a reactive amine for condensation with biotin. In a second method, biotin is attached to the 3' end by RNA ligase.
Zuckermann, et al., Nucleic Acids Research 15(13): 5305-5321 (1987) describe nucleosides modified to incorporate a linker arm having a disulfide link. The disulfide link is used to attach the modified nucleoside to a solid support. The nucleoside is then able to undergo oligonucleotide synthesis according to the phosphoramidite or phosphotriester methods. Following synthesis, the disulfide is cleaved and the free sulfhydryl group may be used to attach a fluorescent reporter to the probe.
Nelson, et al., Nucleic Acids Research 17(18): 7187-7194 (1989) describe a control pore glass solid support having incorporated therein a multifunctional agent having both a masked amino group and a protected hydroxyl group. The hydroxyl group can be used for standard synthesis of oligonucleotides as is known in the art. The protected amino group can be used after synthesis and cleavage of the oligonucleotide for coupling to a reporter molecule. This approach is very much like the commercially available 3' Amine-On CPG.TM. (Clontech, Palo Alto, Calif.)
Each of the above methods have been used successfully to incorporate detectable marker compounds into 3' end positions of oligonucleotides. However, none is readily adaptable to automated synthesis such that the label moiety can be added to the oligonucleotide prior to cleavage from the support. In each case, the oligonucleotide must undergo one or more additional steps to couple the label to the cleaved oligonucleotide. This is undesirable if commercial quantities of labeled oligonucleotides are to be produced by automated synthesis.
Accordingly, the present invention seeks to overcome the problems associated with prior methods, and to provide a solid support and a method for 3' end labeling of oligonucleotides which is amenable to automated chemical synthesis. A method for preparing such a novel support is also described.